Antifungal compositions

ABSTRACT

The present invention relates to new antifungal compositions and their use in the treatment of agricultural products.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a §371 National Stage Application ofPCT/EP2012/052091, filed Feb. 8, 2012, which claims priority to EuropeanApplication No. 11153831.0, filed Feb. 9, 2011.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention discloses new antimicrobial compositions tocontrol plant diseases and to prevent microbial spoilage of crops.

2. Description of Related Art

It is estimated that about 25% of the world crop production is lost dueto microbial spoilage, of which spoilage by fungi is by far the mostimportant cause. Not only from an economical point of view, but alsofrom a humane point of view it is of great importance to preventspoilage of food products. After all, in many parts of the world peoplesuffer from hunger.

Success in combating plant and crop diseases and in reducing the damagethey cause to yields and quality depends greatly on the timelyapplication of fungicides. The prolonged and frequent use of manyfungicides such as e.g. benzamidazoles has contributed to reduce theireffectiveness thanks to the development of phenomena of resistance.

Recently, new fungicides of other chemical families such as e.g.anilinopyrimidines have become commercially available (see WO 03/011030,FR 2 828 065 A1 and WO 2005/074684). Although these fungicides haveshown activity against fungi, spoilage problems still occur. Moreover,several studies have shown that more and more fungi acquire resistanceagainst these fungicides too (see Moyano et al. (2004), Kanetis et al.(2008), Xiao et al. (2011)). Furthermore, many of the currently usedfungicides have the disadvantage of being dangerous for the health ofexposed persons and the environment.

For many decades, fungicides have also been used to prevent fungalgrowth on food products such as cheeses and sausages. EP 0 986 965 A1describes the use of imazalil sulphate to treat cheeses and sausages andprotect them against fungal growth. U.S. Pat. No. 5,597,598 describesthe combined treatment of food products with two acids and the polyenemacrolide antimycotic natamycin. This natural preservative, which isproduced by fermentation using Streptomyces natalensis, is widely usedthroughout the world as a food preservative and has a long history ofsafe use in the food industry. It is very effective against all knownfood spoilage fungi. Although natamycin has been applied for many yearsin e.g. the cheese industry, up to now development of resistant fungalspecies was never observed.

Consequently, it can be concluded that there is a severe need for moreeffective, more environmental friendly, lower-toxicity and less harmfulantimicrobial compositions, e.g. antifungal compositions, for thetreatment of fungal growth in and on plants and crops.

SUMMARY

The present invention solves the problem by providing a new synergisticantimicrobial, e.g. antifungal, composition comprising a polyeneantifungal compound and at least one antifungal compound from the familyof anilinopyrimidines. As used herein, the term “synergistic” means thatthe combined effect of the antifungal compounds when used in combinationis greater than their additive effects when used individually.

In general, synergistic activity of two active ingredients can be testedin for example the analysis of variance model using the treatmentinteraction stratum (see Slinker, 1998). Relative efficacy can becalculated by means of the following formula: ((value of evolutionstatus of untreated control−value of evolution status ofcomposition)/(value of evolution status of untreated control))*100. Aninteraction coefficient can then be calculated by means of the followingformula: ((relative efficacy of combination compound A+compoundB)/(relative efficacy of compound A+relative efficacy of compoundB))*100. An interaction coefficient larger than 100 indicates synergybetween the compounds.

Alternatively, synergy can be calculated as follows: the antifungalactivity (in %) of the individual active ingredients can be determinedby calculating the reduction in mould growth observed on productstreated with the active ingredients in comparison to the mould growth onproducts treated with a control composition. The expected antifungalactivity (E in %) of the combined antifungal composition comprising bothactive ingredients can be calculated according to the Colby equation(Colby, 1967): E=X+Y−[(X·Y)/100], wherein X and Y are the observedantifungal activities (in %) of the individual active ingredients X andY, respectively. If the observed antifungal activity (O in %) of thecombination exceeds the expected antifungal activity (E in %) of thecombination and the synergy factor O/E is thus >1.0, the combinedapplication of the active ingredients leads to a synergistic antifungaleffect.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

In an embodiment of the invention, the at least one antifungal compoundfrom the family of anilinopyrimidines is selected from the groupconsisting of cyprodinil, mepanipyrim and pyrimethanil. In an embodimentthe compositions may also contain two or more different antifungalcompounds from the family of anilinopyrimidines. It is to be understoodthat derivatives of antifungal compounds from the family ofanilinopyrimidines including, but not limited to, salts or solvates ofantifungal compounds from the family of anilinopyrimidines or modifiedforms of antifungal compounds from the family of anilinopyrimidines mayalso be applied in the compositions of the invention. Examples ofcommercial products containing anilinopyrimidines such as pyrimethanilare the products with the brand name Mythos® (pyrimethanil), Scala®(pyrimethanil), Siganex® (pyrimethanil) or Walabi® (pyrimethanil andchlorthalonil). Said commercial products can be incorporated in thepresent invention.

In an embodiment the polyene antifungal compound is selected from thegroup consisting of natamycin, nystatin, amphotericin B, trienin,etruscomycin, filipin, chainin, dermostatin, lymphosarcin, candicidin,aureofungin A, aureofungin B, hamycin A, hamycin B and lucensomycin. Ina preferred embodiment the polyene antifungal compound is natamycin. Inan embodiment the compositions may also contain two or more differentpolyene antifungal compounds. It is to be understood that derivatives ofpolyene antifungal compounds including, but not limited to, salts orsolvates of polyene antifungal compounds or modified forms of polyeneantifungal compounds may also be applied in the compositions of theinvention. Examples of commercial products containing natamycin are theproducts with the brand name Delvocid®. Such products are produced byDSM Food Specialties (The Netherlands) and may be solids containing e.g.50% (w/w) natamycin or liquids comprising between e.g. 2-50% (w/v)natamycin. Said commercial products can be incorporated in thecompositions of the invention.

The composition of the present invention generally comprises from about0.005 g/l to about 100 g/l and preferably from about 0.01 g/l to about50 g/l of a polyene antifungal compound. Preferably, the amount is from0.01 g/l to 3 g/l.

The composition of the present invention generally comprises from about0.0001 g/l to about 2000 g/l and preferably from about 0.0005 g/l toabout 1500 g/l of an antifungal compound from the family ofanilinopyrimidines. More preferably, the amount is from 0.001 g/l to1000 g/l.

In an embodiment the composition of the present invention furthercomprises at least one additional compound selected from the groupconsisting of a sticking agent, a carrier, a colouring agent, aprotective colloid, an adhesive, a herbicide, a fertilizer, a thickeningagent, a sequestering agent, a thixotropic agent, a surfactant, afurther antimicrobial compound, a detergent, a preservative, a spreadingagent, a filler, a spray oil, a flow additive, a mineral substance, asolvent, a dispersant, an emulsifier, a wetting agent, a stabiliser, anantifoaming agent, a buffering agent, an UV-absorber and an antioxidant.A further antimicrobial antifungal compound may be an antifungalcompound (e.g. imazalil, thiabendazole or chlorthalonil) or a compoundto combat insects, nematodes, mites and/or bacteria. Of course, thecompositions according to the invention may also comprise two or more ofany of the above additional compounds. Any of the above mentionedadditional compounds may also be combined with the polyene antifungalcompound and/or the at least one antifungal compound from the family ofanilinopyrimidines in case the antifungal compounds are appliedseparately. In an embodiment the additional compounds are additivesacceptable for the specific use, e.g. food, feed, medicine, cosmetics oragriculture. Additional compounds suitable for use in food, feed,medicine, cosmetics or agriculture are known to the person skilled inthe art.

In a specific embodiment the further antimicrobial compound is a naturalcrop protection compound belonging to the group of phosphites, e.g.KH₂PO₃ or K₂HPO₃ or a mixture of both phosphite salts. Phosphitecontaining compounds as used herein means compounds comprising aphosphite group, i.e. PO₃ (in the form of e.g. H₂PO₃ ⁻, HPO₃ ²⁻ or PO₃³) or any compound which allows the release of a phosphite ion includingcompounds such as phosphorous acid and phosphonic acid as well asderivatives thereof such as esters and/or alkali metal or alkaline earthmetal salts thereof. In case the compositions of the present inventioncomprise a polyene antifungal compound (e.g. natamycin) and at least onephosphite containing compound, they preferably comprise 0.1 g or lesslignosulphonate, more preferably 0.1 g or less polyphenol, per grampolyene antifungal compound. Preferably, they comprise 0.01 g or lesslignosulphonate, more preferably 0.01 g or less polyphenol, per grampolyene antifungal compound. In particular, they are free oflignosulphonate and preferably free of polyphenol. Suitable examples ofphosphite containing compounds are phosphorous acid and its (alkalimetal or alkaline earth metal) salts such as potassium phosphites e.g.KH₂PO₃ and K₂HPO₃, sodium phosphites and ammonium phosphites, and(C₁-C₄) alkyl esters of phosphorous acid and their salts such asaluminum ethyl phosphite (fosetyl-Al), calcium ethyl phosphite,magnesium isopropyl phosphite, magnesium isobutyl phosphite, magnesiumsec-butyl phosphite and aluminum N-butyl phosphite. Of course, mixturesof phosphite containing compounds are also encompassed. A mixture ofe.g. KH₂PO₃ and K₂HPO₃ can easily be obtained by e.g. adding KOH orK₂CO₃ to a final pH of 5.0-6.0 to a KH₂PO₃ solution. As indicated above,precursor-type compounds which in the crop or plant are metabolized intophosphite compounds can also be included in the compositions of thepresent invention. Examples are phosphonates such as thefosetyl-aluminium complex. In e.g. a crop or plant the ethyl phosphonatepart of this molecule is metabolized into a phosphite. An example ofsuch a compound in the commercial ethyl hydrogen phosphonate productcalled Aliette® (Bayer, Germany). The ratio of phosphite to natamycin(in weight) in the compositions is in general between 2:1 to 500:1(w/w), preferably between 3:1 to 300:1 (w/w) and more preferably between5:1 to 200:1 (w/w).

Compositions according to the invention may have a pH of from 1 to 10,preferably of from 2 to 9, more preferably of from 3 to 8 and mostpreferably of from 4 to 7. They may be solid, e.g. powder compositions,or may be liquid. The compositions of the present invention can beaqueous or non-aqueous ready-to-use compositions, but may also beaqueous or non-aqueous concentrated compositions/suspensions or stockcompositions, suspensions and/or solutions which before use have to bediluted with a suitable diluent such as water or a buffer system.Alternatively, the compositions of the invention can also be used toprepare coating emulsions. The compositions of the present invention canalso have the form of concentrated dry products such as e.g. powders,granulates and tablets. They can be used to prepare compositions forimmersion or spraying of products such as agricultural productsincluding plants, crops, vegetables and/or fruits. Of course, the aboveis also applicable when the polyene antifungal compound and the at leastone antifungal compound from the family of anilinopyrimidines areapplied as separate compositions.

In a further aspect the invention relates to a kit comprising a polyeneantifungal compound and at least one antifungal compound from the familyof anilinopyrimidines. The polyene antifungal compound and the at leastone antifungal compound from the family of anilinopyrimidines may bepresent in two separate packages, e.g. containers. The components of thekit may be either in dry form or liquid form in the package. Ifnecessary, the kit may comprise instructions for dissolving thecompounds. In addition, the kit may contain instructions for applyingthe compounds.

In a further aspect the invention pertains to a method for protecting aproduct against fungi by treating the agricultural product with apolyene antifungal compound and at least one antifungal compound fromthe family of anilinopyrimidines. In addition, the product can betreated with other antifungal and/or antimicrobial compounds eitherprior to, concomitant with or after treatment of the products with thepolyene antifungal compound and the at least one antifungal compoundfrom the family of anilinopyrimidines. The product may be treated bysequential application of the polyene antifungal compound and the atleast one antifungal compound from the family of anilinopyrimidines orvice versa. Alternatively, the product may be treated by simultaneousapplication of the polyene antifungal compound and the at least oneantifungal compound from the family of anilinopyrimidines. In case ofsimultaneous application, the compounds can be present in differentcompositions that are applied simultaneously or the compounds may bepresent in a single composition. In yet another embodiment the productmay be treated by separate or alternate modes of applying the antifungalcompounds. In an embodiment the invention is directed to a process forthe treatment of products by applying the polyene antifungal compoundand the at least one antifungal compound from the family ofanilinopyrimidines to the products. By applying the compounds fungalgrowth on or in the products can be prevented. In other words, thecompounds protect the products from fungal growth and/or from fungalinfection and/or from fungal spoilage. The compounds can also be used totreat products that have been infected with a fungus. By applying thecompounds the disease development due to fungi on or in these productscan be slowed down, stopped or the products may even be cured from thedisease. In an embodiment of the invention the products are treated witha composition or kit according to the invention. In an embodiment theproduct is a food, feed, pharmaceutical, cosmetic or agriculturalproduct. In a preferred embodiment the product is an agriculturalproduct.

The polyene antifungal compound and the at least one antifungal compoundfrom the family of anilinopyrimidines, the compositions according to theinvention and the kits according to the invention can be applied to theproducts by spraying. Other methods suitable for applying thesecompounds, compositions and kits in liquid form to the products are alsoa part of the present invention. These include, but are not limited to,dipping, watering, drenching, introduction into a dump tank, vaporizing,atomizing, fogging, fumigating, painting, brushing, dusting, foaming,spreading-on, packaging and coating (e.g. by means of wax orelectrostatically). In addition, the antifungal compounds may also beinjected into the soil. Spraying applications using automatic systemsare known to reduce the labour costs and are cost-effective. Methods andequipment well-known to a person skilled in the art can be used for thatpurpose. The compositions according to the invention can be regularlysprayed, when the risk of infection is high. When the risk of infectionis lower spray intervals may be longer. Depending on the type ofapplication, the amount of polyene antifungal compound applied may varyfrom 5 ppm to 10,000 ppm, preferably from 10 ppm to 5,000 ppm and mostpreferably from 20 to 1,000 ppm. Depending on the type of application,the amount of the at least one antifungal compound from the family ofanilinopyrimidines applied may vary from 10 ppm to 5,000 ppm, preferablyfrom 20 ppm to 3,000 ppm and most preferably from 50 to 1,000 ppm.

In a specific embodiment the agricultural product can be treatedpost-harvest. By using a polyene antifungal compound and the at leastone antifungal compound from the family of anilinopyrimidines thecontrol of post-harvest and/or storage diseases is achieved for a longperiod of time to allow transport of the harvested agricultural productover long distances and under various storage conditions with differentcontrolled atmosphere systems in respect of temperature and humidity.Post-harvest storage disorders are e.g. lenticel spots, scorch,senescent breakdown, bitter pit, scald, water core, browning, vascularbreakdown, CO₂ injury, CO₂ or O₂ deficiency, and softening. Fungaldiseases may be caused for example by the following fungi:Mycosphaerella spp., Mycosphaerella musae, Mycosphaerella fragariae,Mycosphaerella citri; Mucor spp., e.g. Mucor piriformis; Monilinia spp.,e.g. Monilinia fructigena, Monilinia laxa; Phomopsis spp., Phomopsisnatalensis; Colletotrichum spp., e.g. Colletotrichum musae,Colletotrichum gloeosporioides, Colletotrichum coccodes; Verticilliumspp., e.g. Verticillium theobromae; Nigrospora spp.; Botrytis spp., e.g.Botrytis cinerea; Dipodia spp., e.g. Dipodia citri; Pezicula spp.;Alternaria spp., e.g. Alternaria citri, Alternaria alternata; Septoriaspp., e.g. Septoria depressa; Venturia spp., e.g. Venturia inaequalis,Venturia pyrina; Rhizopus spp., e.g. Rhizopus stolonifer, Rhizopusoryzae; Glomerella spp., e.g. Glomerella cingulata; Sclerotinia spp.,e.g. Sclerotinia fruiticola; Ceratocystis spp., e.g. Ceratocystisparadoxa; Fusarium spp., e.g. Fusarium semitectum, Fusarium moniliforme,Fusarium solani, Fusarium oxysporum; Cladosporium spp., e.g.Cladosporium fulvum, Cladosporium cladosporioides, Cladosporiumcucumerinum, Cladosporium musae; Penicillium spp., e.g. Penicilliumfuniculosum, Penicillium expansum, Penicillium digitatum, Penicilliumitalicum; Phytophthora spp., e.g. Phytophthora citrophthora,Phytophthora fragariae, Phytophthora cactorum, Phytophthora parasitica;Phacydiopycnis spp., e.g. Phacydiopycnis malirum; Gloeosporium spp.,e.g. Gloeosporium album, Gloeosporium perennans, Gloeosporiumfructigenum, Gloeosporium singulata; Geotrichum spp., e.g. Geotrichumcandidum; Phlyctaena spp., e.g. Phlyctaena vagabunda; Cylindrocarponspp., e.g. Cylindrocarpon mali; Stemphyllium spp., e.g. Stemphylliumvesicarium; Thielaviopsis spp., e.g. Thielaviopsis paradoxy; Aspergillusspp., e.g. Aspergillus niger, Aspergillus carbonarius; Nectria spp.,e.g. Nectria galligena; Cercospora spp., e.g. Cercospora angreci,Cercospora apii, Cercospora atrofiliformis, Cercospora musae, Cercosporazeaemaydis.

Another aspect of the present invention relates to the use of a polyeneantifungal compound and at least one antifungal compound from the familyof anilinopyrimidines to protect a product against fungi. As indicatedabove, the compounds may be used, e.g. applied, sequentially orsimultaneously. In an embodiment the invention relates to a use, whereina composition or kit according to the invention is applied to theproduct. In an embodiment the product is a food, feed, pharmaceutical,cosmetic or agricultural product. In a preferred embodiment the productis an agricultural product.

A further aspect of the invention is directed to a product treated witha polyene antifungal compound and at least one antifungal compound fromthe family of anilinopyrimidines. In an embodiment the product istreated with a composition or kit according to the invention. Theinvention is therefore directed to a product comprising a polyeneantifungal compound and at least one antifungal compound from the familyof anilinopyrimidines. The treated products may comprise a polyeneantifungal compound and at least one antifungal compound from the familyof anilinopyrimidines on their surface and/or inside the product.Alternatively, the treated products may comprise a coating comprisingthese compounds. In an embodiment the treated products comprise from0.000001 to 200 mg/dm², preferably 0.00001 to 100 mg/dm², morepreferably from 0.00005 to 10 mg/dm² of the polyene antifungal compoundon their surface. In a further embodiment they comprise from 0.000001 to200 mg/dm², preferably 0.00001 to 100 mg/dm², more preferably from0.00005 to 10 mg/dm² of the at least one antifungal compound from thefamily of anilinopyrimidines on their surface. In an embodiment theproduct is a food, feed, pharmaceutical, cosmetic or agriculturalproduct. In a preferred embodiment the product is an agriculturalproduct.

The term “food products” as used herein is to be understood in a verybroad sense and includes, but is not limited to, cheese, cream cheese,shredded cheese, cottage cheese processed cheese, sour cream, driedfermented meat product including salamis and other sausages, wine, beer,yoghurt, juice and other beverages, salad dressing, cottage cheesedressing, dips, bakery products and bakery fillings, surface glazes andicing, spreads, pizza toppings, confectionery and confectioneryfillings, olives, olive brine, olive oil, juices, tomato purees andpaste, condiments, and fruit pulp and the like food products.

The term “feed products” as used herein is also to be understood in avery broad sense and includes, but is not limited to, pet food, broilerfeed, etc.

The term “pharmaceutical product” as used herein is also to beunderstood in a very broad sense and includes products comprising anactive molecule such as a drug, agent, or pharmaceutical compound andoptionally a pharmaceutically acceptable excipient, i.e. any inertsubstance that is combined with the active molecule for preparing anagreeable or convenient dosage form.

The term “cosmetic product” as used herein is also to be understood in avery broad sense and includes products that are used for protecting ortreating horny tissues such as skin and lips, hair and nails from dryingby preventing transpiration of moisture thereof and further conditioningthe tissues as well as giving good appearance to these tissues. Productscontemplated by the term “cosmetic product” include, but are not limitedto, moisturizers, personal cleansing products, occlusive drug deliverypatches, nail polish, powders, wipes, hair conditioners, skin treatmentemulsions, shaving creams and the like.

The term “agricultural products” as used herein is also to be understoodin a very broad sense and includes, but is not limited to, cereals, e.g.wheat, barley, rye, oats, rice, sorghum and the like; beets, e.g. sugarbeet and fodder beet; pome and stone fruit and berries, e.g. apples,pears, plums, apricots, peaches, almonds, cherries, strawberries,raspberries and blackberries; leguminous plants, e.g. beans, lentils,peas, soy beans; oleaginous plants, e.g. rape, mustard, poppy, olive,sunflower, coconut, castor-oil plant, cocoa, ground-nuts; cucurbitaceae,e.g. pumpkins, gherkins, melons, cucumbers, squashes, aubergines;fibrous plants, e.g. cotton, flax, hemp, jute; citrus fruit, e.g.oranges, lemons, grapefruits, mandarins, limes; tropical fruit, e.g.papayas, passion fruit, mangos, carambolas, pineapples, bananas, kiwis;vegetables, e.g. spinach, lettuce, asparagus, brassicaceae such ascabbages and turnips, carrots, onions, tomatoes, potatoes,seed-potatoes, hot and sweet peppers; laurel-like plants, e.g. avocado,cinnamon, camphor tree; or products such as maize, tobacco, nuts,coffee, sugarcane, tea, grapevines, hops, rubber plants, as well asornamental plants, e.g. cut flowers, roses, tulips, lilies, narcissus,crocuses, hyacinths, dahlias, gerbera, carnations, fuchsias,chrysanthemums, and flower bulbs, shrubs, deciduous trees and evergreentrees such as conifers, plants and trees in greenhouses. It includes,but is not limited to, plants and their parts, fruits, seeds, cuttings,cultivars, grafts, bulbs, tubers, root-tubers, rootstocks, cut flowersand vegetables.

A method for preparing a composition as described herein is anotheraspect of the present invention. The method comprises adding a polyeneantifungal compound to at least one antifungal compound from the familyof anilinopyrimidines. The compounds may for instance be addedseparately to an aqueous composition and mixed, followed, if necessary,by adjustment of the pH, viscosity, etc. If added separately, some orall of the separate compounds may be in powder form, but alternativelysome or all may also be in liquid form. The compounds may for instancealso be added to one another in powder form and mixed to obtain apowdered composition. The powdered composition may then be added to anaqueous composition.

Example 1 Pre-Harvest Application

Leaves of banana plants are inoculated with fungi. As a controlnon-inoculated leaves are also included. Next, a defined part of theleaves are treated with composition 1 (natamycin), composition 2(pyrimethanil) or composition 3 (natamycin+pyrimethanil). Eachcomposition is applied by spraying. Untreated leaves are also included(untreated control).

The obtained results show that the compositions of the present inventionprotect banana plants from fungal growth and further demonstrate thatthe compositions of the present invention show a synergisticallyenhanced activity compared to the activity of the active compounds whenapplied individually.

Example 2 Post-Harvest Application

Bananas are injured according to the method described by de Lapeyre deBellaire and Dubois (1987). Bananas are wounded using a cork borerfollowed by contamination with fungal spores. After incubation forseveral hours at room temperature, the bananas are dipped in one of thefollowing compositions: a) no treatment (control 1), b) dipped in water(control 2), c) dipped in natamycin, d) dipped in pyrimethanil, e)dipped in natamycin+pyrimethanil. After this treatment the bananas areincubated in closed boxes at 21° C. at elevated humidity. Each day thebananas are judged visually on fungal development.

The results show that the composition comprising natamycin andpyrimethanil protects bananas better against fungi than natamycin orpyrimethanil alone. Surprisingly, the combined application of natamycinand pyrimethanil leads to a strong synergistic reduction in infection.

Example 3 Treatment of Bananas

Four organic, unripe (green) bananas were used per treatment. The peelof each banana was wounded thrice using a cork borer according to themethod described by de Lapeyre de Bellaire and Dubois (1987).Subsequently, each wound was inoculated with 15 μl of a Fusariumproliferatum suspension containing 1×10⁵ of spores/ml. After incubationfor 4 hours at 20° C., each banana wound was treated with 100 μl of afreshly prepared aqueous antifungal composition comprising either 500ppm natamycin (DSM Food Specialties, Delft, The Netherlands), 1000 ppmcyprodinil or both. In addition, the antifungal compositions comprised1.00% (w/w) methylhydroxyethylcellulose (MHEC), 0.40% (w/w) xanthan gum,0.20% (w/w) anti-foaming agent, 0.30% (w/w) citric acid, 0.39% (w/w)lactic acid and 0.11% (w/w) potassium sorbate. A composition withoutnatamycin or cyprodinil was used as control. The treated, unripe bananaswere incubated in a closed box in the dark at 20° C. and a relative airhumidity of 95%, which was obtained in the presence of a saturatedNa₂HPO₄ aqueous solution. During the first 20 days of incubation, a ripe(yellow) banana was included in the closed box to elevate the ethylenegas level and thus induce ripening of the treated, unripe bananas.

During incubation, the degree of mould growth on the bananas wasassessed in a twofold manner: (i) the number of moulded wounds per totalof 12 wounds was counted; and (ii) the antifungal activity (in %) of theindividual active ingredients was determined by calculating thereduction in mould growth observed on the banana wounds treated with theantifungal composition in comparison to the mould growth on the bananawounds treated with the control composition. The expected antifungalactivity (E in %) of the combined antifungal composition comprising bothactive ingredients was calculated according to the Colby equation(Colby, 1967):E=X+Y−[(X·Y)/100]wherein X and Y are the observed antifungal activities (in %) of theindividual active ingredients X and Y, respectively. If the observedantifungal activity (0 in %) of the combination exceeds the expectedantifungal activity (E in %) of the combination and the synergy factorO/E is thus >1.0, the combined application of the active ingredientsleads to a synergistic antifungal effect.

The results in Table 1 (number of moulded wounds per total of 12 wounds)and Table 2 (antifungal activity) clearly demonstrate that theantifungal composition comprising both 500 ppm natamycin and 1000 ppmcyprodinil protected bananas better against mould growth than natamycinor cyprodinil alone.

After 23 days of incubation, all 12 wounds treated with the controlcomposition or with cyprodinil alone showed mould growth, whereas 9 ofthe 12 wounds treated with natamycin alone were moulded. However, mouldgrowth was observed only for 6 of the 12 wounds treated with thecomposition comprising both natamycin and cyprodinil (see Table 1).

After 27 days of incubation, all 12 wounds treated with either thecontrol composition, natamycin alone or cyprodinil alone showed mouldgrowth. However, mould growth was observed only for 7 of the 12 woundstreated with the composition comprising both natamycin and cyprodinil(see Table 1). Furthermore, the observed antifungal activity of thecomposition comprising both natamycin and cyprodinil was 13% higher thanthe expected antifungal activity and a synergy factor of >1.0 wasobtained (see Table 2).

After 39 days of incubation, the observed antifungal activity of thecomposition comprising both natamycin and cyprodinil was 6% higher thanthe expected antifungal activity and a synergy factor of >1.0 wasobtained (see Table 2).

Hence, the combination of 500 ppm natamycin and 1000 ppm cyprodinil hassynergistic antifungal activity on bananas.

Example 4 Treatment of Bananas

The experiment was conducted as described in Example 3, except for thefact that each inoculated banana wound was treated with 100 μl of afreshly prepared aqueous antifungal composition comprising either 250ppm natamycin (DSM Food Specialties, Delft, The Netherlands), 500 ppmcyprodinil or both. The degree of mould growth on the banana wounds wasassessed according to the two methods described in Example 3.

The results in Table 3 (number of moulded wounds per total of 12 wounds)and Table 4 (antifungal activity) reveal that the antifungal compositioncomprising 250 ppm natamycin as well as 500 ppm cyprodinil was superiorto the compositions comprising either natamycin alone or cyprodinilalone in reducing mould growth on bananas.

After 18 and 21 days of incubation, all 12 wounds treated with eitherthe control composition, natamycin alone or cyprodinil alone showedmould growth. However, when treated with the composition comprising bothnatamycin and cyprodinil, only 6 of the 12 wounds were moulded after 18days and 7 of the 12 wounds were moulded after 21 days (see Table 3).

Furthermore, after 21 days of incubation the actually observedantifungal activity of the composition comprising both natamycin andcyprodinil was 23% higher than the expected antifungal activity, whichresulted in a synergy factor of 1.5 (see Table 4).

Moreover, after 32 and 34 days of incubation, the observed antifungalactivity of the composition comprising both natamycin and cyprodinil was30% higher than the expected antifungal activity. Consequently, thesynergy factors far exceeded 1.0 (see Table 4).

In conclusion, the results of this example clearly demonstrate that theantifungal activity of the combination of 250 ppm natamycin and 500 ppmcyprodinil when applied on bananas is highly synergistic.

Example 5 Treatment of Bananas

The experiment was conducted as described in Example 3, except for thefact that each inoculated banana wound was treated with 100 μl of afreshly prepared aqueous antifungal composition comprising either 50 ppmnatamycin (DSM Food Specialties, Delft, The Netherlands), 250 ppmcyprodinil or both. The antifungal activity (in %) of the individual andcombined active ingredients on the banana wounds was determinedaccording to the method described in Example 3.

The results (see Table 5) show that the combined antifungal compositioncomprising 50 ppm natamycin and 250 ppm cyprodinil protected bananasmore effectively against mould growth than the compositions comprisingnatamycin or cyprodinil alone.

After 23, 29, 32 and 36 days of incubation, the observed antifungalactivity of the composition comprising both natamycin and cyprodinil was10 to 21% higher than the expected antifungal activity. The synergyfactor ranged from 2.3 to >10 after 23 and 36 days of incubation,respectively.

Thus, the combined application of 50 ppm natamycin and 250 ppmcyprodinil leads to a surprisingly strong synergistic reduction in mouldgrowth on bananas.

Example 6 Treatment of bananas

The experiment was conducted as described in Example 3, except for thefact that each inoculated banana wound was treated with 100 μl of afreshly prepared aqueous antifungal composition comprising either 250ppm natamycin (DSM Food Specialties, Delft, The Netherlands), 500 ppmpyrimethanil or both. A composition without natamycin or pyrimethanilwas used as control. The antifungal activity (in %) of the individualand combined active ingredients on the banana wounds was determinedaccording to the method described in Example 3.

The results (see Table 6) clearly demonstrate that the combinedantifungal composition comprising 250 ppm natamycin and 500 ppmpyrimethanil had a much stronger antifungal activity on bananas than thecompositions comprising natamycin or pyrimethanil alone.

After 23, 29, 32 and 34 days of incubation, the observed antifungalactivity of the composition comprising both natamycin and pyrimethanilwas 4 to 11% higher than the expected antifungal activity. As a result,all synergy factors exceeded 1.0 and increased from 1.5 on day 23 to >11on day 34.

Hence, the antifungal activity of the combination of 250 ppm natamycinand 500 ppm pyrimethanil is strongly synergistic when applied onbananas.

Example 7 Treatment of Strawberries

Twelve fresh, organic strawberries were used per treatment. Eachstrawberry was wounded with a 0.5 mm long cut and each wound wasinoculated with 10 μl of a Botrytis cinerea suspension containing 1×10⁵of spores/ml. After a 2-hour incubation period at 20° C., eachstrawberry was dipped individually for 1 minute in a freshly preparedaqueous antifungal composition comprising either 250 ppm natamycin (DSMFood Specialties, Delft, The Netherlands), 500 ppm pyrimethanil or both.The antifungal composition also comprised 1.00% (w/w)methylhydroxyethylcellulose (MHEC), 0.40% (w/w) xanthan gum, 0.20% (w/w)anti-foaming agent, 0.30% (w/w) citric acid, 0.39% (w/w) lactic acid and0.11% (w/w) potassium sorbate. A composition without natamycin orpyrimethanil was used as control.

The treated strawberries were incubated for three days in a closed boxin the dark at 20° C. and assessed daily on mould growth. The antifungalactivity (in %) of the individual active ingredients was determined bycalculating the reduction in mould growth observed on the strawberriestreated with the antifungal composition in comparison to the mouldgrowth on the strawberries treated with the control composition. Theexpected antifungal activity (E in %) of the combined antifungalcomposition comprising both active ingredients was calculated accordingto the Colby equation (Colby, 1967):E=X+Y−[(X·Y)/100]wherein X and Y are the observed antifungal activities (in %) of theindividual active ingredients X and Y, respectively. If the observedantifungal activity (O in %) of the combination exceeds the expectedantifungal activity (E in %) of the combination and the synergy factorO/E is thus >1.0, the combined application of the active ingredientsleads to a synergistic antifungal effect.

The results (see Table 7) show that the antifungal compositioncomprising 250 ppm natamycin and 500 ppm pyrimethanil protectedstrawberries much better against mould growth than natamycin orpyrimethanil alone.

After 1, 2 and 3 days of incubation, the observed antifungal activity ofthe combined composition comprising natamycin and pyrimethanil wasrespectively 25, 21 and 14% higher than the expected antifungalactivity. The corresponding synergy factors ranged from 1.6 to 2.0.

Hence, the combined application of 250 ppm natamycin and 500 ppmpyrimethanil leads to a synergistic reduction in mould growth onstrawberries.

Example 8 Treatment of Mandarins

Ten fresh, organic mandarins were used per treatment. The peel of eachmandarin was wounded once using a cork borer according to the methoddescribed by de Lapeyre de Bellaire and Dubois (1987). Subsequently,each wound was inoculated with 10 μl of a Penicillium italicumsuspension containing 1×10⁴ of spores/ml. After incubation for 2 hoursat 20° C., the mandarins were dipped individually for 1 minute in afreshly prepared aqueous antifungal composition comprising either 250ppm natamycin (DSM Food Specialties, Delft, The Netherlands), 500 ppmcyprodinil or both. In addition, the antifungal composition comprised3.1% (w/w) beeswax, 0.76% (w/w) glycerol, 0.66% (w/w) polyoxyethylenesorbitan monostearate (Tween 60), 0.03% (w/w)methylhydroxyethylcellulose (MHEC), 0.02% (w/w) xanthan gum, 0.02% (w/w)anti-foaming agent, 0.15% (w/w) citric acid and 0.01% (w/w) potassiumsorbate. A composition without natamycin or cyprodinil was used ascontrol.

The treated mandarins were incubated in a closed box in the dark at 20°C. and assessed on mould growth after 16, 19, 22 and 26 days ofincubation. The antifungal activity (in %) of the individual activeingredients was determined by calculating the reduction in mould growthobserved on the mandarins treated with the antifungal composition incomparison to the mould growth on the mandarins treated with the controlcomposition. The expected antifungal activity (E in %) of the combinedantifungal composition comprising both active ingredients was calculatedaccording to the Colby equation (Colby, 1967):E=X+Y−[(X·Y)/100]wherein X and Y are the observed antifungal activities (in %) of theindividual active ingredients X and Y, respectively. If the observedantifungal activity (O in %) of the combination exceeds the expectedantifungal activity (E in %) of the combination and the synergy factorO/E is thus >1.0, the combined application of the active ingredientsleads to a synergistic antifungal effect.

The results (see Table 8) clearly demonstrate that the antifungalcomposition comprising 250 ppm natamycin and 500 ppm cyprodinil wassuperior to the compositions comprising natamycin or cyprodinil alone inpreventing mould growth on mandarins.

After 16, 19, 22 and 26 days of incubation, the observed antifungalactivity of the composition comprising both natamycin and cyprodinil was31 to 62% higher than the expected antifungal activity. Consequently,all synergy factors exceeded 1.0 and increased from 1.5 on day 16 to 4.3on day 26.

Hence, the results of this example clearly demonstrate the synergisticantifungal activity between 250 ppm natamycin and 500 ppm cyprodinil onmandarins.

Example 9 Treatment of Mandarins

The experiment was conducted as described in Example 8, except for thefact that each wounded, inoculated mandarin was dipped individually for1 minute in a freshly prepared aqueous antifungal composition comprisingeither 500 ppm natamycin (DSM Food Specialties, Delft, The Netherlands),1000 ppm pyrimethanil or both. A composition without natamycin orpyrimethanil was used as control. The antifungal activity (in %) of theindividual and combined active ingredients on the banana wounds wasassessed after 22 and 26 days of incubation according to the methoddescribed in Example 8.

The results in Table 9 prove that the antifungal composition comprising500 ppm natamycin and 1000 ppm pyrimethanil reduced mould growth onmandarins more effectively than natamycin or pyrimethanil alone.

After 22 and 26 days of incubation, the observed antifungal activity ofthe composition comprising both natamycin and pyrimethanil exceeded theexpected antifungal activity with respectively 11 and 13% and synergyfactors >1.0 were obtained.

It can therefore be concluded that the combined application of 500 ppmnatamycin and 1000 ppm pyrimethanil leads to a synergistic reduction inmould growth on mandarins.

Example 10 Treatment of Mandarins

The experiment was conducted as described in Example 8, except for thefact that each wounded, inoculated mandarin was dipped individually for1 minute in a freshly prepared aqueous antifungal composition comprisingeither 250 ppm natamycin (DSM Food Specialties, Delft, The Netherlands),500 ppm pyrimethanil or both. A composition without natamycin orpyrimethanil was used as control. The antifungal activity (in %) of theindividual and combined active ingredients on the banana wounds wasassessed after 19, 22 and 26 days of incubation according to the methoddescribed in Example 8. The results (see Table 10) reveal that theantifungal composition comprising 500 ppm natamycin and 1000 ppmpyrimethanil had a stronger antifungal activity on mandarins than thecompositions comprising natamycin or pyrimethanil alone.

After 19, 22 and 26 days of incubation, the observed antifungal activityof the composition comprising both natamycin and pyrimethanil exceededthe expected antifungal activity with 15, 21 and 29%, respectively, andsynergy factors >1.0 were obtained.

In conclusion, the antifungal activity of the active ingredientcombination of 250 ppm natamycin and 500 ppm pyrimethanil is synergisticwhen applied on mandarins.

Example 11 In Vitro Antifungal Activity

To show synergistic activity of the combination of natamycin withcyprodinil or pyrimethanil, an in vitro assay was conducted using96-well microtiter plates. The following compositions were tested:

Control (no active ingredient),

0.63 ppm of natamycin (DSM Food Specialties, Delft, The Netherlands),

1.25 ppm of natamycin,

10 ppm of cyprodinil,

20 ppm of cyprodinil,

17.5 ppm of pyrimethanil,

30 ppm of pyrimethanil,

0.63 ppm of natamycin+20 ppm of cyprodinil,

0.63 ppm of natamycin+30 ppm of pyrimethanil,

1.25 ppm of natamycin+10 ppm of cyprodinil

1.25 ppm of natamycin+17.5 ppm of pyrimethanil.

After filling each well with 92 μl of PCB medium, natamycin andcyprodinil or pyrimethanil were added from separate stock solutionsprepared in PCB medium or methanol, which resulted in an intermediatevolume of 100 μl per well. Subsequently, 100 μl of a Botrytis cinereasuspension prepared in PCB medium was used to inoculated each well with2.5×10³ spores/ml. Each well thus contained a final volume of 200 μl and<1% of methanol, which did not affect fungal growth (data not shown).

After incubation of the microtiter plates for 5 days at 25° C., the invitro antifungal activity (%) of the individual active ingredients wasassessed by calculating the reduction in mould growth observed in thepresence of the active ingredient in comparison to the mould growthobserved in the absence of the active ingredient. The expectedantifungal activity (E in %) of the active ingredient combination wascalculated according to the Colby equation (Colby, 1967):E=X+Y−[(X·Y)/100]wherein X and Y are the observed antifungal activities (in %) of theindividual active ingredients X and Y, respectively. If the observedantifungal activity (O in %) of the combination exceeds the expectedantifungal activity (E in %) of the combination and the resultingsynergy factor O/E is thus >1.0, the combined application of the activeingredients leads to a synergistic antifungal effect.

The results (see Table 11) demonstrate that the combinationsnatamycin+cyprodinil and natamycin+pyrimethanil had much strongerantifungal activities against Botrytis cinerea than natamycin,cyprodinil and pyrimethanil individually. Moreover, the observedantifungal activities of the combinations natamycin+cyprodinil andnatamycin+pyrimethanil were 50 to 100% higher than the expectedantifungal activities, resulting in synergy factors far above 1.0.

Hence, the combined application of natamycin and cyprodinil as well asthe combined application of natamycin and pyrimethanil synergisticallyinhibit growth of Botrytis cinerea.

Example 12 In Vitro Antifungal Activity

The experiment was conducted as described in Example 11, except for thefact that the following compositions were tested:

Control (no active ingredient),

1.25 ppm of natamycin (DSM Food Specialties, Delft, The Netherlands),

125 ppm of cyprodinil,

1.25 ppm of natamycin+125 ppm of cyprodinil,

2.5 ppm of natamycin,

275 ppm of pyrimethanil,

2.5 ppm of natamycin+275 ppm of pyrimethanil.

Furthermore, Fusarium proliferatum was used for inoculation. Theantifungal activity (in %) of the individual and combined activeingredients was determined according to the method described in Example11.

The results (see Table 12) reveal that the active ingredientcombinations natamycin+cyprodinil and natamycin+pyrimethanil inhibitgrowth of Fusarium proliferatum more effectively than natamycin,cyprodinil and pyrimethanil individually. Moreover, the observedantifungal activities of the active ingredient combinationsnatamycin+cyprodinil and natamycin+pyrimethanil were 100% higher thanthe expected antifungal activities, resulting in synergy factors farabove 1.0.

Hence, the combined application of natamycin and cyprodinil and thecombined application of natamycin and pyrimethanil both lead to a strongsynergistic reduction in the growth of Fusarium proliferatum.

TABLE 1 Number of moulded wounds on bananas incubated at 20° C. aftertreatment with compositions comprising either 500 ppm natamycin, 1000ppm cyprodinil or both. Incubation Number of moulded wounds/ Antifungalcomposition time (days) total number of 12 wounds Control 23 12/12Natamycin 500 ppm  9/12 Cyprodinil 1000 ppm 12/12 Natamycin 500 ppm + 6/12 cyprodinil 1000 ppm Control 27 12/12 Natamycin 500 ppm 12/12Cyprodinil 1000 ppm 12/12 Natamycin 500 ppm +  7/12 cyprodinil 1000 ppm

TABLE 2 Antifungal activity (%) of compositions comprising either 500ppm natamycin, 1000 ppm cyprodinil or both on bananas incubated at 20°C. Observed Expected Incubation antifungal antifungal Synergy timeactivity O activity E factor Antifungal composition (days) (%) (%) O/EControl 27 0 — — Natamycin 500 ppm 63 — — Cyprodinil 1000 ppm 18 — —Natamycin 500 ppm + 83 70 1.2 cyprodinil 1000 ppm Control 39 0 — —Natamycin 500 ppm 11 — — Cyprodinil 1000 ppm 0 — — Natamycin 500 ppm +17 11 1.6 cyprodinil 1000 ppm

TABLE 3 Number of moulded wounds on bananas incubated at 20° C. aftertreatment with compositions comprising either 250 ppm natamycin, 500 ppmcyprodinil or both. Incubation Number of moulded wounds/ Antifungalcomposition time (days) total number of 12 wounds Control 18 12/12Natamycin 250 ppm 12/12 Cyprodinil 500 ppm 12/12 Natamycin 250 ppm + 6/12 cyprodinil 500 ppm Control 21 12/12 Natamycin 250 ppm 12/12Cyprodinil 500 ppm 12/12 Natamycin 250 ppm +  7/12 cyprodinil 500 ppm

TABLE 4 Antifungal activity (%) of compositions comprising either 250ppm natamycin, 500 ppm cyprodinil or both on bananas incubated at 20° C.Observed Expected antifungal antifungal Synergy Incubation activity Oactivity E factor Antifungal composition time (days) (%) (%) O/E Control21 0 — — Natamycin 250 ppm 10 — — Cyprodinil 500 ppm 35 — — Natamycin250 ppm + 64 41  1.5 cyprodinil 500 ppm Control 32 0 — — Natamycin 250ppm 2 — — Cyprodinil 500 ppm 3 — — Natamycin 250 ppm + 36 5 7.0cyprodinil 500 ppm Control 34 0 — — Natamycin 250 ppm 0 — — Cyprodinil500 ppm 0 — — Natamycin 250 ppm + 30 0 >30 cyprodinil 500 ppm

TABLE 5 Antifungal activity (%) of compositions comprising either 50 ppmnatamycin, 250 ppm cyprodinil or both on bananas incubated at 20° C.Observed Expected antifungal antifungal Synergy Incubation activity Oactivity E factor Antifungal composition time (days) (%) (%) O/E Control23 0 — — Natamycin 50 ppm 12 — — Cyprodinil 250 ppm 0 — — Natamycin 50ppm + 28 12  2.3 cyprodinil 250 ppm Control 29 0 — — Natamycin 50 ppm 4— — Cyprodinil 250 ppm 0 — — Natamycin 50 ppm + 25 4 6.3 cyprodinil 250ppm Control 32 0 — — Natamycin 50 ppm 1 — — Cyprodinil 250 ppm 0 — —Natamycin 50 ppm + 19 1 19 cyprodinil 250 ppm Control 36 0 — — Natamycin50 ppm 0 — — Cyprodinil 250 ppm 0 — — Natamycin 50 ppm + 10 0 >10cyprodinil 250 ppm

TABLE 6 Antifungal activity (%) of compositions comprising either 250ppm natamycin, 500 ppm pyrimethanil or both on bananas incubated at 20°C. Observed Expected antifungal antifungal Synergy Incubation activity Oactivity E factor Antifungal composition time (days) (%) (%) O/E Control23 0 — — Natamycin 250 ppm 19 — — Pyrimethanil 500 ppm 0 — — Natamycin250 ppm + 28 19  1.5 pyrimethanil 500 ppm Control 29 0 — — Natamycin 250ppm 9 — — Pyrimethanil 500 ppm 0 — — Natamycin 250 ppm + 13 9 1.5pyrimethanil 500 ppm Control 32 0 — — Natamycin 250 ppm 2 — —Pyrimethanil 500 ppm 0 — — Natamycin 250 ppm + 10 2 5.0 pyrimethanil 500ppm Control 34 0 — — Natamycin 250 ppm 0 — — Pyrimethanil 500 ppm 0 — —Natamycin 250 ppm + 11 0 >11 pyrimethanil 500 ppm

TABLE 7 Antifungal activity (%) of compositions comprising either 250ppm natamycin, 500 ppm pyrimethanil or both on strawberries incubated at20° C. Observed Expected antifungal antifungal Synergy Incubationactivity O activity E factor Antifungal composition time (days) (%) (%)O/E Control 1 0 — — Natamycin 250 ppm 25 — — Pyrimethanil 500 ppm 0 — —Natamycin 250 ppm + 50 25 2.0 pyrimethanil 500 ppm Control 2 0 — —Natamycin 250 ppm 31 — — Pyrimethanil 500 ppm 0 — — Natamycin 250 ppm +52 31 1.7 pyrimethanil 500 ppm Control 3 0 — — Natamycin 250 ppm 24 — —Pyrimethanil 500 ppm 0 — — Natamycin 250 ppm + 38 24 1.6 pyrimethanil500 ppm

TABLE 8 Antifungal activity (%) of compositions comprising either 250ppm natamycin, 500 ppm cyprodinil or both on mandarins incubated at 20°C. Observed Expected antifungal antifungal Synergy Incubation activity Oactivity E factor Antifungal composition time (days) (%) (%) O/E Control16 0 — — Natamycin 250 ppm 15 — — Cyprodinil 500 ppm 60 — — Natamycin250 ppm + 97 66 1.5 cyprodinil 500 ppm Control 19 0 — — Natamycin 250ppm 5 — — Cyprodinil 500 ppm 38 — — Natamycin 250 ppm + 92 41 2.3cyprodinil 500 ppm Control 22 0 — — Natamycin 250 ppm 5 — — Cyprodinil500 ppm 20 — — Natamycin 250 ppm + 86 24 3.5 cyprodinil 500 ppm Control26 0 — — Natamycin 250 ppm 5 — — Cyprodinil 500 ppm 13 — — Natamycin 250ppm + 73 17 4.3 cyprodinil 500 ppm

TABLE 9 Antifungal activity (%) of compositions comprising either 500ppm natamycin, 1000 ppm pyrimethanil or both on mandarins incubated at20° C. Observed Expected antifungal antifungal Synergy Incubationactivity O activity E factor Antifungal composition time (days) (%) (%)O/E Control 22 0 — — Natamycin 500 ppm 37 — — Pyrimethanil 1000 ppm 82 —— Natamycin 500 ppm + 100 89 1.1 pyrimethanil 1000 ppm Control 26 0 — —Natamycin 500 ppm 30 — — Pyrimethanil 1000 ppm 79 — — Natamycin 500ppm + 98 85 1.1 pyrimethanil 1000 ppm

TABLE 10 Antifungal activity (%) of compositions comprising either 250ppm natamycin, 500 ppm pyrimethanil or both on mandarins incubated at20° C. Observed Expected antifungal antifungal Synergy Incubationactivity O activity E factor Antifungal composition time (days) (%) (%)O/E Control 19 0 — — Natamycin 250 ppm 5 — — Pyrimethanil 500 ppm 84 — —Natamycin 250 ppm + 100 85 1.2 pyrimethanil 500 ppm Control 22 0 — —Natamycin 250 ppm 5 — — Pyrimethanil 500 ppm 74 — — Natamycin 250 ppm +96 75 1.3 pyrimethanil 500 ppm Control 26 0 — — Natamycin 250 ppm 5 — —Pyrimethanil 500 ppm 65 — — Natamycin 250 ppm + 96 67 1.4 pyrimethanil500 ppm

TABLE 11 In vitro antifungal activity (%) of natamycin in combinationwith cyprodinil or pyrimethanil against Botrytis cinerea after 5 days ofincubation at 25° C. Observed Expected Synergy antifungal antifungalfactor Antifungal composition activity O (%) activity E (%) O/E Control0 — — Natamycin 0.63 ppm 0 — — Cyprodinil 20 ppm 0 — — Pyrimethanil 30ppm 50 — — Natamycin 0.63 ppm + 100 0 >100 cyprodinil 20 ppm Natamycin0.63 ppm + 100 50 2 pyrimethanil 30 ppm Control 0 — — Natamycin 1.25 ppm0 — — Cyprodinil 10 ppm 0 — — Pyrimethanil 17.5 ppm 50 — — Natamycin1.25 ppm + 100 0 >100 cyprodinil 10 ppm Natamycin 1.25 ppm + 100 50 2pyrimethanil 17.5 ppm

TABLE 12 In vitro antifungal activity (%) of natamycin in combinationwith cyprodinil or pyrimethanil against Fusarium proliferatum after 5days of incubation at 25° C. Observed Expected Synergy antifungalantifungal factor Antifungal composition activity O (%) activity E (%)O/E Control 0 — — Natamycin 1.25 ppm 0 — — Cyprodinil 125 ppm 0 — —Natamycin 1.25 ppm + 100 0 >100 cyprodinil 125 ppm Control 0 — —Natamycin 2.5 ppm 0 — — Pyrimethanil 275 ppm 0 — — Natamycin 2.5 ppm +100 0 >100 pyrimethanil 275 ppm

REFERENCES

-   Colby S R (1967), Calculating synergistic and antagonistic responses    of herbicide combination. Weeds 15: 20-22.-   Kanetis L, Förster H, Jones C A, Borkovich K A and Adaskaveg J E    (2008),-   Characterization of genetic and biochemical mechanisms of    fludioxonil and pyrimethanil resistance in field isolates of    Penicillium digitatum. Phytopathology 98: 2005-214.-   Lapeyre de Bellaire de L and Dubois C (1987), Distribution of    Thiabendazole-Resistant Colletotrichum musae Isolates from    Guadeloupe Banana Plantations. Plant disease 81:1378-1383.-   Moyano C, Gomez V and Melgarejo P (2004), Resistance to pyrimethanil    and other fungicides in Botrytis cinerea populations collected on    vegetable crops in Spain. J. Phytopathology 152: 484-490.-   Slinker B K (1998), The Statistics of Synergism. Journal of Mol. and    Cell. Cardiology 30:723-731.-   Xiao C L, Kim Y K and Boal R J (2011), First report of occurrence of    pyrimethanil resistance in Penicillium expansu, from stored apples    in Washington State. Plant Disease 95: 72.

The invention claimed is:
 1. A composition comprising natamycin and atleast one antifungal compound selected from the group consisting ofcyprodinil and pyrimethanil.
 2. A composition according to claim 1,wherein the composition further comprises at least one additionalcompound selected from the group consisting of a sticking agent, acarrier, a colouring agent, a protective colloid, an adhesive, aherbicide, a fertilizer, a thickening agent, a sequestering agent, athixotropic agent, a surfactant, a further antimicrobial compound, adetergent, a preservative, a spreading agent, a filler, a spray oil, aflow additive, a mineral substance, a solvent, a dispersant, anemulsifier, a wetting agent, a stabiliser, an antifoaming agent, abuffering agent, an UV-absorber and an antioxidant.
 3. A compositionaccording to claim 1, wherein the amount of natamycin is in a range from0.005 g/l to about 100 g/l and the amount of the at least one antifungalcompound is in a range from about 0.0001 g/l to about 2000 g/l.
 4. Thecomposition of claim 1 which comprises natamycin and cyprodinil.
 5. Thecomposition of claim 4 wherein the amount of natamycin is in the rangeof 0.63 to 500 ppm, and the amount of cyprodinil is in the range of 10ppm to 1000 ppm.
 6. The composition of claim 1 which comprises natamycinand pyrimethanil.
 7. The composition of claim 6 wherein the amount ofnatamycin is in the range of 0.63 to 500 ppm, and the amount ofpyrimethanil is in the range of 17.5 ppm to 1000 ppm.
 8. A kitcomprising natamycin and at least one antifungal compound selected fromthe group consisting of cyprodinil and pyrimethanil.
 9. A productcomprising natamycin and at least one antifungal compound selected fromthe group consisting of cyprodinil and pyrimethanil.
 10. A productaccording to claim 9, wherein said product is selected from the groupconsisting of a food product, a feed product, a pharmaceutical product,a cosmetic product and an agricultural product.
 11. A product accordingto claim 10, wherein said product is an agricultural product.
 12. Amethod for protecting a product against fungi comprising treating theproduct with natamycin and at least one antifungal compound selectedfrom the group consisting of cyprodinil and pyrimethanil.
 13. A methodaccording to claim 12, wherein said product is selected from the groupconsisting of a food product, a feed product, a pharmaceutical product,a cosmetic product and an agricultural product.
 14. A method accordingto claim 13, wherein said product is an agricultural product.
 15. Amethod according to claim 14, wherein said product is treatedpost-harvest.
 16. A method according to claim 12, wherein said productis treated with a composition comprising natamycin and at least oneantifungal compound selected from the group consisting of cyprodinil andpyrimethanil.
 17. A method according to claim 16, wherein said productis selected from the group consisting of a food product, a feed product,a pharmaceutical product, a cosmetic product and an agriculturalproduct.